Suspended ultra-sound induced microbubble cavitation imaging

ABSTRACT

An ultrasonic imaging technique is disclosed which uses microbubbles as echo contrast agents. In general the method employs maitenance of an ultrasound signal while the contrast agent is intravenously injected into a mammal. Once all the contrast agent has been injected and transmission of the signal is suspended for a period of time sufficient for the microbubbles perfuse the organ of interest. Transmission of the ultrasound signal is then resumed and peak contrast images are obtained which rival more complicated imaging procedures such as nuclear resonance imaging.

This application is a continuation of application Ser. No. 08/721,507,filed Sep. 26, 1996, now U.S. Pat. No. 5,740,807 which is a continuationof 08/439,619, filed May 12, 1995, issued as U.S. Pat. No. 5,560,364Oct. 1, 1996, the disclosure of which is hereby incorprated byreference.

BACKGROUND OF THE INVENTION

Ultrasonic imaging is used as a diagnostic tool to aid in therapeuticprocedures. It is based on the principle that waves of sound energy canbe focused upon an area of interest and reflected to produce an image.Generally, an ultrasonic transducer is placed on a body surfaceoverlying the area to be imaged, and ultrasonic energy, produced bygenerating and receiving sound waves is transmitted. The ultrasonicenergy is reflected back to the transducer where it is translated intoan ultrasonic image. The amount and characteristics of the reflectedenergy depend upon the acoustic properties of the tissues, and contrastagents which are echogenic are preferentially used to create ultrasonicenergy in an area of interest and improve the image received.

In ultrasound imaging, videotape images obtained following contrastinjection are digitized, allowing the gray scale to be quantified from 1to 225 gray scale units for 30 cardiac cycles. The contrast intensity isplotted on the vertical axis against time on the horizontal axis. Thepeak videointensity (corrected for baseline intensity) is determined asthe highest point on the time intensity curve.

For a discussion of contrast echographic instrumentation, see, forexample, De Jong N, "Acoustic properties of ultrasound contrast agents",CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAG (1993), pages 120 et seq.

Contrast echocardiography has been used to delineate intracardiacstructures, assess valvular competence, and demonstrate intracardiacshunts. Myocardial contrast echocardiography (MCE) has been used tomeasure coronary blood flow reserve in humans. MCE has been found to bea safe and useful technique for evaluating relative changes inmyocardial perfusion and delineating areas at risk.

A multiplicity of potential ultrasonic imaging agents has been reportedfor contrast echocardiography. No such agent routinely attains visuallydiscernible myocardial uptake following peripheral intravenousinjection. Although there have been many reports of transpulmonarytransmission of ultrasound contrast agents following intravenousinjection and despite the fact that myocardial opacification onechocardiogram can be produced by left sided injection of such contrastagents, visualization of myocardial contrast has not been achieved byintravenous administration of sonicated microbubbles.

Most recently, sonicated albumin and sonicated dextrose/albumin havebeen shown to produce variable degrees of left ventricular chamberultrasound contrast following intravenous injection. (See Villanueva etal. Circulation 85:1557-1564, 1992; Lin et al. Int J Card Imaging8:53-6, 1992; Feinstein et al. J Am Coll Cardiol 16:316-224, 1990;Keller et al. Am Heart J 114:570-575, 1987; and Shapiro et al. J Am CollCardiol 16:1603-1607, 1990). The microbubbles of these contrast agentsare small (4-6 microns) and are capable of swift transpulmonary passage.However, visually discernible myocardial uptake of such microbubblesfollowing peripheral intravenous injection has not been possible becauseof the rapid diffusion of blood soluble oxygen and nitrogen inside themicrobubble into the blood which consequently loses its ultrasoundreflective properties (e.g., see Porter et al. J Am Soc EchocardSupplement 7:S1, May 1994, and Weyman AE: Principles and Practice ofEchocardiography, Malvern, Pennsylvania: Lea & Febiger, 1994; pp.302-26.)

Despite recent advances in contrast agents comprising injectable gasencapsulated microbubbles, several problems remain for adequatedetection and visualization of the organ of interest. The bubbles areplagued with filtration by capillaries, diffusion of gas to the liquidmedium, and lack of concentration at the organ of interest due todilution.

Attempts to solve these problems have led to studies of acousticvelocity of media containing gas bubbles, second harmonic emission, andresonance frequency. These studies to date have met with littleimprovement in contrast visualization.

It is an object of the present invention to provide a safe, simple andeffective method to visualize and improve an ultrasound image followinginjection of gas-filled microbubbles.

Other objects of the invention will be apparent from the description ofthe invention which follows.

SUMMARY OF THE INVENTION

According to the invention, after introduction of a microbubble contrastagent the ultrasound transmission is suspended to allow the contrastagent to perfuse the organ of interest and is then resumed for astriking improvement in contrast. Use of this method results in up toten times improvement in contrast, decreases acoustic shadowing andallows for reduction in the amount of contrast agent necessary, therebyreducing toxicity and improving safety.

DETAILED DESCRIPTION OF THE INVENTION

The primary problem in contrast ultrasound imaging is the detection andquantification of perfusion in the anatomical organs of interest, suchas heart, kidney, liver etc. The most success to date has been withusing contrast gas-filled microbubbles which are injected intravenouslyinto the bloodstream.

Often, however, the intravenously injected bolus of microbubbles getsdiluted in concentration as it travels to the targeted organ ofinterest, making detection difficult if not impossible. One of theprimary problems in echocardiography is the detection of myocardialperfusion. More importantly, as the bolus flows through the right-sideof the heart and passes through the lungs, microbubbles greater than 8microns in size are filtered out by the pulmonary capillaries.

Further, as the bubbles flow into the left-side of the heart, some ofthe bubbles are destroyed during the course of the heart cycle as theyare subjected to the fairly high systolic pressures in the leftventricle (LV). Some of the bubbles disappear by the simple process ofdiffusion of gas into the surrounding liquid medium.

Finally, the bubbles in the ventricle strongly attenuate the incidentultrasound field and cast an acoustic shadow on the distal myocardium.Hence, there is a trade-off between increasing the dose for improvedmicrobubble detection sensitivity and the need for minimizing theacoustical shadowing of the myocardial beds. All of this is exacerbatedby the fact that only about 4% of the total blood volume in the LVcavity enters the coronary circulation. As a net result, only a smallnumber of microbubbles of small size (from the original bolus injection)traverse the myocardial vasculature.

This invention alleviates these concerns by allowing for detection ofvery small amounts of contrast agent, improving contrast by as much asten times and can be used in echocardiography as well as for renal orhepatic imaging. The invention (suspended ultrasound induced cavitationimaging) comprises a modification of standard ultrasound imagingtechniques and can be used with any ultrasound imaging device incombination with a microbubble contrast agent.

In this method the ultrasound signal is operating (i.e. the transucer isheld in position over the organ of interest, for example the heart) uponintroduction of the contrast agent. The contrast agent is then injectedperipherally, in an amount sufficient for visual detection of the organof interest. Standard amounts of microbubble contrast agent may be used,from about 0.04 ml/kg to about 0.08 ml/kg for humans however it will beseen that much smaller amounts will work equally as well with the methodof the invention, as little as 0.001 ml/kg to 0.0025 ml/kg. The doserange is patient specific as large patients may require slightly higherdoses to produce equivalent left ventricular contrast. Standardmethodology for contrast echocardiography are described in Weyman,Arthur E. "Principles and Practice of Echocardiography" Lea and Fibiger,Malvern, Pa. (1994 2d Ed).

After injection the transmission of the ultrasound signal isinterrupted, or suspended for a period of time sufficient for thecontrast agent to perfuse the organ of interest. The time period willgenerally vary according to the organ of interest. It must only be longenough for some of the agent to have reached the organ of interest. Forthe heart this would be the time sufficient for the contrast agent toreach the myocardium. This is approximately about 10-120 seconds,preferrably around 20-30 seconds and should not vary significantly frompatient to patient.

The length of time for other organs or for any particular patient may beeasily ascertained by conducting standard ultrasound imaging where thetransmission is not interrupted and timing the period from injection toperfusion of the organ of interest.

Once the microbubbles have reached the organ of interest or myocardiumfor echocardiography, the transmission of the ultrasound signal isresumed and peak myocardial contrast images are obtained.

In echocardiography there will be a uniform opacification of the entiremyocardium. Within a few seconds, the intensity of the entire myocardiumuniformly drops to the original (dark) intensity level. Regions of themyocardial bed that are devoid of blood flow and contrast remain dark atall times. Hence, it is easy to differentiate between normally perfusedand abnormally perfused myocardial regions.

While not wishing to be bound by any theory, it is postulated that theobserved phenomenon arises as a result of the transient response of thecontrast microbubbles when subjected to a driving ultrasonic field. Theultrasound signal causes the microbubbles to compress, or cavitate. Whenthe microbubbles are injected, they become compressed, as they encounterthe ultrasonic field. When the ultrasound signal is suspended, thetransmit power into the anatomy is shut off. In the absence of aradiating field, the microbubbles in the myocardium or other organ, growin size by a finite amount. Some may even coalesce to form largerscatterers.

The reflection of the ultrasound signal will then be enhanced by thelarger size of the microbubbles. This is because the backscatteredultrasound energy is directly proportional to the number of scatterersin the region of interest and varies as the sixth power of the radius ofthe scatterer.

When the ultrasound system is resumed, the transmit power into theanatomy is enabled and will generate the image scan on the display. Peakcontrast images are obtained at this point, before the ultrasound signalbegins to cavitate the microbubbles once again. As the ultrasound fieldbuilds up from zero to steady state, the pressure on the microbubblesincreases causing them to diminish in size once again. This transientdecrease in scatterer size causes the resultant transient decrease inmyocardial intensity.

It is postulated that another explanation for the increase in intensityupon resuming ultrasound transmission may be that the impressedultrasound field sputters the microbubbles and any coalesced microbubbleaggregates into numerous microbubbles thereby effectively increasing thenumber of scatterers and thereby causing the increased myocardialopacification. The contrast is so improved that the image producedrivals that of more complicated imaging such as nuclear resonanceimaging.

The main advantage of this invention is that it allows for sensitivedetection and quantification of perfusion at the target organ in anon-invasive mode using a very small dose of contrast microbubbleswithout producing acoustical shadows. Because of the small dose, patientsafety is enhanced and the cost is also minimized.

For most ultrasound imaging, the contrast agent is formulated in apharmaceutically effective dosage form for peripheral administration tothe host to be imaged. Generally such host is a human subject althoughother mammalian hosts, such as canine or equine can be imagedeffectively. In a most preferred embodiment the contrast agent is asonicated mixture of commercially available albumin (human), USP,solution (generally supplied as 5% or a 25%, by weight, sterile aqueoussolutions), and commercially available dextrose, USP, for intravenousadministration are employed. This mixture is sonicated under ambientconditions, i.e., room air, temperature and pressure, and is perfusedwith perfluorocarbon or other commercially available inert gas (99.9% byweight) during sonication.

In a preferred embodiment the invention uses a microbubble contrastagent wherein the microbubbles are stabilized by a filmogenic,de-naturable protein coating. Suitable proteins include naturallyoccurring proteins such as albumin, human gamma-globulin, humanapotransferrin, Beta-lactose, and urease. The invention preferablyemploys a naturally occurring protein, but synthetic proteins may alsobe used. Particularly preferred is human serum albumin.

Although intravenous echo contrast agents made from sonicatedmicrobubbles are known (e.g., ALBUNEX, Molecular Biosystems, Inc.) andcan be employed in this invention, it is preferred to use a sonicatedaqueous solution containing a mixture of a pharmaceutically acceptablesaccharide, e.g., dextrose, and a protein, e.g., albumin. Generally,sonication is performed in an air atmosphere. In an especially preferredembodiment, dextrose, which is readily available in pharmaceuticallyacceptable dosage forms, is the preferred saccharide and human serumalbumin is the preferred protein. The preferred embodiment would includea two-fold to eight-fold dilution of 5%-50% by weight of dextrose and a2%-10% by weight of human serum albumin. Exemplary of other saccharidesolutions of this invention are an aqueous monosaccharide solution (e.g.having the formula C₆ H₆ O₁₂, such as, the hexoses, dextrose orfructose, or mixtures thereof), aqueous disaccharide solution (e.g.,having the formula C₁₂ H₂₂ O₁₁, such as sucrose, lactose or maltose, ormixture thereof), or aqueous polysaccharide solution (e.g., solublestarches having the formula (C₆ H₁₀ ₅)_(n), wherein n is a whole integerbetween about 20 and about 200, such as amylose or dextran, or mixturesthereof. Sonication by ultrasonic energy causes cavitation within thedextrose-albumin solution at sites of particulate matter or gas in thefluid. These cavitation sites eventually resonate and produce smallmicrobubbles (about 4 to about 7 microns in size) which arenon-collapsing and stable. In general, sonication conditions whichproduce concentrations of greater than about 4×10⁸ m of between about 5and about 6 micron microbubbles are preferred.

The mean microbubble size of sonicated dextrose albumin ranges frombetween about 5 to about 6 microns. This is a good size as it has beenobserved that microbubble radius decreases as a function of time in astill liquid due to a diffusion gradient present between the internaland external gases of the microbubble. An increase in microbubble sizehas a significant effect on the persistence of a microbubble withinblood. It must also be of a size sufficient for transpulmonary passage.It must have a mean diameter of less than 10 microns and greater 0.1microns. Since the size of albumin microbubbles is ideal (between 5 and6 microns) for transpulmonary passage, the major reason for thesignificant loss in left ventricular and myocardial videointensityproduced following intravenous injection of albumin coated microbubblesis due to the significant diffusion of gases within the microbubblefollowing intravenous injection during transit to the left ventricularcavity. Sonicated dextrose albumin enhanced with an inert gas such asperfluorocarbon gas, having a lower blood solubility than air, and amolecular weight of greater than 100 grams/mole, produces asignificantly higher left ventricular and myocardial videointensity thansonicated albumin alone.

Because of high surface tension, the concentration of nitrogen andoxygen gas within the microbubble is much higher than that in blood, andthus there is a rapid diffusion of this gas into the blood streamfollowing intravenous injection. Wible et al. (Circulation, 88:I-401,1993) have demonstrated that this diffusion process can be acceleratedif one decreased the partial pressure of nitrogen within the bloodstream by decreasing the inhaled fraction of nitrogen. This lowerexternal concentration of nitrogen results in loss of the leftventricular videointensity produced by the same intravenous injection ofsonicated albumin while inhaling room air. It has also been observedthat oxygen rapidly diffuses out of gas bubbles into human blood (SeeYang et al., J Biomech 3:275, 1971).

Both nitrogen and oxygen diffuse rapidly across these concentrationgradients, but nitrogen appears to dissolve more slowly than oxygen intoblood. Since nitrogen is the major component of air, decreasing theconcentration gradient for nitrogen across the microbubble improves leftventricular and myocardial videointensity following intravenousinjection. Exposing the microbubbles to a non-toxic gas having a lowerblood solubility and/or microbubble diffusivity than that of nitrogenand having a gas density of greater than about 0.300 lb/ft³ duringsonication increases the size and stability of the microbubbles insonicated dextrose albumin, while lowering the solubility anddiffusivity of the microbubbles in blood. Suitable gases are those whichare gas at 37° C. and which are nontoxic. Insoluble gases useful forcontrast agents include but are not limited to prefluorocarbon gasessuch as perfluoromethane, perfluoroethane, perfluoropropane,perflouorobutane, perfluoropentane etc., or sulfur hexafluoride. In apreferred embodiment the gas is perfluoropropane (C₃ F₈) orperfluorobutane (C₄ F₁₀). The perfluorocarbon gas content of themicrobubbles is sufficient to lower microbubble gas solubility anddiffusivity in vivo in blood. Generally, the minimum amount of insolublegas in the microbubbles which is effective is that amount which resultsin microbubbles which pass reliably through the pulmonary circulationwithout collapse. This is evidenced by opacification of the myocardiumof the left ventricle of the heart following intravenous injection andcan be visually discerned by echocardiography.

In addition to myocardial imaging the contrast agents of this inventionare useful for renal and hepatic imaging. Thus, another embodiment ofthis invention provides a method for myocardial, renal or hepaticopacification. The method preferred involves obtaining an echo contrastagent of this invention, introducing said echo contrast agent into ahost by intravenous injection, and performing an echo contrast study onsaid host using a suitable Doppler or ultrasound echo apparatus asdiscussed more fully hereinafter.

In a most preferred embodiment the contrast agent is aperfluorobutane-enhanced sonicated dextrose albumin solution comprisedof a sonicated three-fold dilution of 5% human serum albumin with 5%dextrose. During sonication, said solution is perfused withperfluoropropane for about 80 seconds, and concomitantly exposed toperfluorobutane or perfluoropropane for at least about 5 seconds duringsonication. This lowers the solubility and diffusivity of themicrobubble gas. The resulting microbubbles are concentrated at roomtemperature for at least about 120±5 minutes, wherein the excesssolution settles in the sonicating syringe. The excess solution isexpelled and the concentrated microbubbles are transferred to a sterilesyringe and injected intravenously into a mammal.

Using the method of the invention in echocardiography will result in ahigher degree of myocardial opacification, endocardial borderdelineation, and enhanced detection of left-sided ultrasound Dopplersignals, upon peripheral venous administration. Additionally, the methodof the invention allow for increased sensitivity as small doses ofmicrobubbles are easily detected, which subsequently enables ultrasonicvisualization of the liver and kidneys following an intravenousinjection.

The following examples are for illustration purposes only and are in noway intended to limit the invention. It will be apparent to those ofskill in the art that other embodiments are practicable and evenintended.

EXAMPLE 1 Preparation of Contrast Agents

Albumin (human) USP, 5% solution (hereinafter referred to as "albumin")and dextrose USP, 5% solution (hereinafter referred to as "dextrose")were obtained from a commercial source. The sonicating system used forsonication was a Heat System Ultrasonic Processor Model XL2020 (HeatSystems Inc., Farmingdale, New York). The 1/2 inch horn transducer was aresonating piezoelectric device. The 1/2 inch sonicating horn tip wassterilized prior to each sonication.

Sonication of Samples

Sixteen milliliter aliquots of albumin diluted 1:3 with dextrose weredrawn up into a 35 cc "Monoject" syringe (Becton Dickinson and Company,Rutherford, N.J.) and sonicated for 80±1 seconds. The "Leur-Lok" of the35 milliliter syringe is then attached to a stopcock. After mixing thedextrose albumin solution by hand for about 7 to about 10 seconds, theplunger was removed from the top of the syringe. The sterile sonicatinghorn was then lowered into the open end of the syringe until at thesurface of the albumin-dextrose solution. The solution was placed at thehorn tip and manually held at this position while continuouslysonicating at a frequency of 20,000 Hz and a power output of 210 W for80±1 seconds to form a stable microbubble solution.

Gas Perfusion of Samples

During sonication, the dextrose albumin mixture was exposed to eitherperfluorobutane or perfluoropropane gas (Commercial Grade, 99.9% byweight). The gas was drawn up into a sterile syringe through a 0.22 μMfilter (Micron Separations Inc., Westborough, Massachusetts) to preventcontamination. During sonication, 5 milliliters of the perfluorocarbongas was manually injected into the solution, over the 80 second timeinterval, through the stopcock so that the microbubbles producedcontained this less soluble gas. The total volume ofperfluorobutane-enhanced sonicated dextrose albumin (BESDA) orperfluoropropane-enhanced sonicated dextrose albumin (PESDA) producedwith this formulation was 25±2 milliliters. These samples were then usedfor intravenous injection.

Microbubble Analysis

Microbubble size and purity was determined using hemocytometry.Microscopic inspection of the microbubbles was performed to determine ifany coalescent microbubbles were present in the solution. Microbubbleconcentration was determined using a Coulter Counter. The contrast agentwas rejected for use if any of the following conditions were present:the mean microbubble size is 4.0 to 6.0 microns; coalesced microbubblesor strands were detected by light microscopy; or the mean microbubbleconcentration was less than 0.8×10⁹ or greater than 1.5×10⁹microbubble/milliliter. The sample was also rejected if the number ofmicrobubbles greater than 10 microns in the sample was greater than 4%.

All samples were stored in 35 milliliter syringes until time ofinjection. All solutions were given within 36 hours of production. Allsamples were prepared in a laminar flow hood.

EXAMPLE 2 Preparation of Open-Chest Dogs

Mongrel dogs of either sex (15-30 kilograms) were anesthetized withsodium pentobarbital (30 milligram per kilogram intravenously),intubated, and ventilated initially with room air using a positivepressure respirator. A left thoracotomy was performed under sterileconditions and the pericardium incised. In addition to a 19 gaugeperipheral intravenous line, eight French Catheters were placed in thefemoral artery and vein for intravenous administration of ultrasoundcontrast agents and pressure monitoring. Through one femoral venoussheath, a 7F balloon-tipped thermodilution catheter was placed in thepulmonary artery using fluoroscopy for determination of pulmonary arterypressure and cardiac output. A 7F pigtail catheter was introduced intothe left ventricle for pressure measurements (left ventricular systolicand end-diastolic pressure) following injection of each ultrasoundcontrast agent.

Following adequate surgical exposure, a 3.5 Megahertz ultrasoundtransducer connected to a commercially available ultrasound scanner(Hewlett Packard Company; Andover, Massachusetts) was placed in a warmwater bath. The bath overlays the anterior epicardial surface. Thetransducer was mounted on a clamp and lowered into the bath. It wasadjusted until an optimal stable short axis view of the left and rightventricle had been obtained at the ventricular mid-papillary musclelevel. These images were then used to assess left ventricular cavity andmyocardial uptake of contrast following intravenous injection.

EXAMPLE 3

Delayed Ultrasound-Induced Cavitation Imaging vs. ConventionalUltrasound Imaging for Echocardiography

To determine if the acoustic shadowing could be decreased or eliminatedwith the method of the invention, four human patients were testedcomparing conventional ultrasound imaging with delayedultrasound-induced cavitation imaging. For this experiment, myocardialcontrast was determined using on-line digitally acquired videointensityobtained from software supplied in conjunction with the commerciallyavailable ultrasound system (Hewlett-Packard Sonos 1500 Phased Arrayimaging system, Hewlett Packard, Andover, Mass.). The peak myocardialvideointensity (PMVI), duration of acoustic shadowing (AS), and percdntof myocardial contrast (MC) were observed. The results are shown inTable 1 below.

Each patient was injected intravenously with 0.0025-0.005 milliliter perkilogram of contrast agent, prepared as in Example 1. The fluorocarbongas agent used was decafluorobutane. For the conventional ultrasoundimaging, the transmission of ultrasound was constant at 2.5-2.7 MHz andimaging was performed continuously throughout the experiment. Peakcontrast images of the myocardium were obtained once the microbubbles ofthe contrast agent reached the myocardium.

For the delayed ultrasound-induced cavitation imaging, the transmissionof ultrasound was maintained at 2.5-2.7 MHz until the patient wasinjected with contrast agent. Upon intravenous injection of contrastagent, the ultrasound transmission was suspended for 20 to 30 seconds.Ultrasound transmission was resumed at 2.5-2.7 MHz once the microbubblesof the contrast agent reached the myocardium. Peak myocardial contrastimages were obtained immediately following commencement of theultrasound transmission and before the microbubbles began to compress.

                                      TABLE 1                                     __________________________________________________________________________    Myocardial Contrast in Ultrasound Imaging                                                Dose of                                                                           Ultrasound                                                                              Acoustic                                                                            Myocardial                                     Imaging                                                                                   Contrast                                                                         Signal                                                                                   Shadowing                                                                          Contrast                                       Modality                                                                            Patient #                                                                           Agent                                                                               Delay Time                                                                       PMVI                                                                               (AS)      (MC)                                      __________________________________________________________________________    UIC* 1) 2.7 MHz                                                                          0.005                                                                             20    19  10 sec                                                                              2+                                                     2) 2.5 MHz                                                                           0.0025                                                                          20         23 sec                                                                              2+                                                  3) 2.5 MHz                                                                           0.0025                                                                          25           8 sec                                                                             1+                                                  4) 2.5 MHz                                                                           0.005                                                                            30        12 sec                                                                              2+                                          CONV**                                                                              1) 2.7 MHz                                                                             0.005                                                                             0        20 sec                                                                              2+                                                2) 2.5 MHz                                                                             0.0025                                                                           0         25 sec                                                                              1+                                                3) 2.5 MHz                                                                             0.0025                                                                           0         20 sec                                                                              1+                                                4) 2.5 MHz                                                                             0.005                                                                             0        30 sec                                                                              2+                                          __________________________________________________________________________     * delayed ultrasound induced cavitation                                       ** conventional ultrasound imaging                                       

The results of this experiment indicate that conventional imagingfollowing intravenous injection of the perfluorocarbon-enhancedsonicated dextrose albumin contrast agent produced a high degree ofacoustic shadowing in the posterior structures of the left ventricularcavity. The delayed ultrasound-induced cavitation imaging decreased theamount of acoustic shadowing by a sufficient amount to enable theimaging of inferior defects of the myocardium. The method of theinvention was also compared to standard myocardial imaging in dogs withan increase in myocardial contrast of ten times that of conventionalmethod. A marked reduction in acoustic shadowing was also seen.

EXAMPLE 4 Effects of Transducer Frequency and Power on CavitationUltrasound Imaging

The effects of transducer frequency and transducer acoustic output wasdemonstrated in the method of the invention using two open chest dogs.

Two dogs received a total of 22 intravenous injections of aperfluoropropane-enhanced sonicated dextrose albumin contrast agent(prepared as in Example 1) at doses between 0.005 and 0.010 millilitersper kilogram body weight. The diagnostic ultrasound signal wastransmitted only after the contrast agent microbubbles had reached themyocardium and peak myocardial videointensity (PMVI) and the duration ofmyocardial contrast (dur-MC) were measured following each injection. Thetransducer frequency and acoustic output were varied between eachinjection. The results are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Effects of Transducer Frequency and                                           Power on Cavitation Ultrasound Imaging                                        Transducer                                                                                                  Duration of                                     Frequency                                                                              Acoustic Output                                                                                       myocardial contrast                          (MHz)        (decibels)                                                                                  PMVI                                                                                (dur-MC)                                     ______________________________________                                        2.0-2.5 158-170       42 ± 6                                                                             5 ± 1                                        2.0-2.5 212                    1 ± 1*. 6                                   2.7-3.5 158-170            37 ± 7                                                                         11 ± 1**                                    2.7-3.5 212                   3 ± 1+-. 7                                   ______________________________________                                         * p < 0.05, compared to other transducer frequencies                          ** p < 0.05, compared to other durations                                 

The results indicate that the best myocardial contrast imaging isobtained when the transducer frequency is low and a low acoustic outputprolongs the duration of this phenomenon.

What is claimed is:
 1. A method of ultrasound imaging of a myocardial,renal, or hepatic tissue which employs an echo contrast agent havingmicrobubbles comprising:introducing said echo contrast agent into ananimal by intravenous injection; compressing the microbubbles using anultrasonic field; temporarily allowing the microbubbles to grow in sizeand thereafter; recompressing the microbubbles in order to visualizesaid tissue.
 2. The method of claim 1 wherein said animal is a dog andsaid step of introducing contrast agent is in an amount of from about0.005 ml/kg to about 0.030 ml/kg.
 3. The method of claim 1 wherein saidanimal is a human and said step of introducing said contrast agent is inan amount of from about 0.001 ml/kg to about 0.0020 ml/kg.
 4. The methodof claim 1 wherein said step of introducing said echo contrast agentcomprises:introducing a contrast agent with perfluorocarbon gas or otherinsoluble gas with a molecular weight of greater than 100 grams/moleencapsulated microbubbles.
 5. The method of claim 4 wherein saidperfluorocarbon gas is selected from the group consisting ofperfluoromethane, perfluorobutane, perfluoroethane, perfluoropropane anda blood insoluble gas with a molecular weight of greater than 100grams/mole.
 6. The method of claim 5 wherein said perfluorocarbon gas isperfluorbutane.
 7. The method of claim 1 wherein said microbubbles areencapsulated by a filmogenic protein.
 8. The method of claim 7 whereinsaid human serum albumin is diluted with dextrose.
 9. The method ofclaim 7 wherein said dilution of human serum albumin with dextrose isthree to one.
 10. The method of claim 7 wherein said human serum albuminis a 5% by weight solution and said dextrose is a 5% by weight solution.11. The method of claim 1 wherein the microbubbles are allowed to growin size for a time period of approximately 10-120 seconds.
 12. Themethod of claim 7 wherein the filmogenic protein is human serum albumin.13. A method of ultrasound imaging of a myocardial, renal, or hepatictissue comprising:preparing an echo contrast agent which comprisesmicrobubbles; introducing said echo contrast agent into an animal;compressing the microbubbles; temporarily allowing the microbubbles togrow in size and thereafter; recompressing the microbubbles in order tovisualize said tissue.
 14. The method of claim 13 wherein themicrobubbles are allowed to grow in size for a period of approximately10 to approximately 30 seconds.
 15. The method of claim 13 wherein saidstep of preparing said echo contrast agent includes the step of:dilutinga solution of 5% by weight albumin with 5% by weight dextrose by 3 to 1to create a mixture; and adding a gas to the mixture to causemicrobubble formation.
 16. The method of claim 15 further comprising thestep of: sonicating said mixture in the presence of a perfluorocarbongas.
 17. The method of claim 16 wherein said prefluorocarbon gas isselected from the group consisting of perfluoropropane andperfluorobutane.
 18. The method of claim 17 wherein said perfluorocarbongas is perfluorobutane.